Anthracycline antibiotics such as doxorubicin and daunorubicin are among the most potent and clinically important antineoplastons. However, their toxicity interferes with their therapeutic use against human malignancy. In addition, tumor cells develop resistance to these antibiotics after repeated treatments. This characteristic of tumor cells is known as multidrug resistance (MDR).
Although a variety of biological mechanisms give rise to MDR, it is usually caused by the overexpression of P-glycoprotein, which is believed to be involved in causing the efflux of drugs from cells, including cancer cells. This mechanism affects not only anthracycline antibiotics but many structurally unrelated anticancer drugs as well.
Theoretically, at least two approaches may be used to avoid MDR. The first approach is to use therapeutics that suppress the expression of P-glycoprotein. Unfortunately, this approach has met with little success. A second approach is to identify cancer therapeutics that do not cause overexpression of P-glycoprotein. Annamycin is one such drug.
Annamycin is an anthracycline derivative that has been proven by in vitro and in vivo studies to have low cardiotoxicity and it does not cause MDR. Even though it does not exhibit MDR, Annamycin's potential as an effective cancer therapeutic has been limited by the lack of a method for producing the drug in pure form or in sufficient quantities for therapeutic use. Known methods of synthesis are technically difficult and cannot be scaled up. Furthermore, the yield of Annamycin that is produced by known synthetic methods is unsatisfactory. See Horton et al., 1984; U.S. Pat. No. 4,537,882.
Using the procedure of Horton et al., 1984 and U.S. Pat. No. 4,537,882, which are specifically incorporated herein by reference, the overall yield of Annamycin from the starting material, 4-demethoxydaunomycinone, is at most, 3%. When the Horton procedure is scaled up, yields are frequently as low as 2%, even when enantiomerically pure starting materials are used. Furthermore, the purity of the final product is only about 80%. These preparations are too impure for therapeutic use, which requires higher purity and detailed knowledge concerning the nature of impurities.
In the procedure of Horton et al. (1984) silylated Annamycin precursor is prepared by the condensation of silylated adriamycinone derivative with 3,4-di-O-acetyl-L-rhamnal. This reaction gives a mixture of two products, one having the undesirable gluco configuration and the other having the desired manno configuration.
In the Horton procedure the reaction product having the manno configuration must be purified away from the gluco form. This is a difficult separation because the two compounds have similar polarities. To separate the compounds, a silica column having a mass that is 100 times that of the sample is required. In addition, the polarity of solvent required to elute the desired product is low. This further reduces the recovery of product from the column. Thus, more than half of the Annamycin precursor is lost because of irreversible adsorption to the silica. Furthermore, silica columns do not completely resolve the manno and gluco species and almost half of the product collected is collected as a mixture of the gluco and manno species.
In the end, the Horton method requires 16 grams (g) of protected adriamycinone derivative, a 5 kilogram (kg) silica gel column and 200 liters (L) of solvent (a toluene/acetone (99.3/0.7) mixture) to produce just 9 g of the silylated 3,4-di-O-acetyl-L-manno adriamycinone intermediate. In the process, about 7 g of the mixed gluco and manno forms is obtained and chromatography of this mixture must be repeated. Ultimately, the overall product yield in this synthetic step ranges from 16% to 40%. In addition to the unacceptably low yields, it is clear that this procedure cannot be scaled up to produce the quantities of Annamycin required for therapeutic use.
Another inefficient step in the synthesis of Annamycin is the step for removing the silyl protecting group from the silylated Annamycin intermediate. In the Horton method, this protecting group is removed by treating the purified manno intermediate with tetrabutylammonium fluoride in a pyridine/THF solution. However, the basic lability of the anthracyclines substantially reduces the yield of product from this reaction and the Annamycin obtained is generally no more than 80% pure.
Yields are decreased further during purification. Recoveries from the silica column used in the final purification are low because Annamycin is highly polar and poorly soluble. The overall yield of the purified product in this step is between 10-15%, which also is too low for commercial viability.
New synthetic methods are needed that can be used to produce Annamycin to a purity of over 95% and in a relatively high yield. To accomplish this, improved methods for preparing the pure silyl derivative of Annamycin are needed. In addition, improved methods for removing the silyl protecting group from the silylated Annamycin precursor that leave the molecule intact are needed, as are improvements to methods for purifying the Annamycin product after the desilylation reaction. A method that includes these improvements could be used to produce large quantities of Annamycin in a sufficient purity for therapeutic use.